Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ
Figure 3
Quantitative analysis of PLP-specific CD4 T cells detected by in situ staining with PLP 139–151 dextramers in EAEmice.
Three paired cerebral sections, each 200 µm thick, were made from brains obtained from EAEmice using vibratome by cutting the sections from the dorsal to the ventral surface with 1.5 mm intervals between pairs. One section from each pair was stained with either PLP 139–151 or TMEV 70–86 (control) dextramers together with anti-CD4. After fixing and mounting, the sections were examined by LSCM (60X), and each inflammatory focus was subjected to a set of ‘Z’ serial images (15 to 25) taken sequentially. In total, 10 to 15 sets of such ‘Z’ serial images representing as many inflammatory foci were acquired from each section. In all the images, cells positive for both PLP 139–151 dextramers and CD4 or CD4 alone were counted using ‘ImageJ’ software. Finally, the cells counted in all the ‘Z’ serial images obtained from all the inflammatory foci were added to obtain the total number per section.